Based on about three decades of research in yeast and vertebrate cells in culture, cell cycle regulation by checkpoints is believed to be of paramount importance in the survival of single cells challenged with genotoxins. The prior art, however, is less clear on whether cell cycle regulation by checkpoints is equally important for the survival of multicellular tissues, organs, and organisms that are similarly challenged.
In eukaryotes, DNA damage checkpoints monitor the state of genomic DNA and delay the progress through the cell division cycle (reviewed in Elledge, Science 274: 1664 (1996) and in Zhou & Elledge, Nature 408: 433 (2000). Components of the signal transduction pathway that constitute the DNA damage checkpoint are well-characterized and include, in mammals, two P13-like kinases, ATM and ATR, and two serine/threonine kinases, CHK1 and CHK2. Homologs of these kinases are found from yeast to worm to fly to human and assume similar roles where examined.
Much of current and recent work on DNA damage checkpoints has revealed in detail the molecular nature of their interface with the cell cycle machinery. For example, delay of G2/M transition in fission yeast is initiated when CHK1 phosphorylates CDC25, a phosphatase that activates cyclin dependent kinase 1 (CDK1). Phosphorylation of CDC25 by CHK1 allows the binding and inactivation of the former by a 14-3-3 protein, thereby keeping CDK1 in the phosphorylated and inactive form. This delays the entry into mitosis (Lopez-Girona et al., Nature 397: 172 (1999); Lopez-Girona et al., Curr. Biol. 11:50 (2001)). In another example, delay of S phase entry is elicited when Chk2 phosphorylates and stabilizes p53, which in turn promotes transcription of p21; p21 inhibits CDK2 to delay G1/S transition (Brugarolas et al., Nature 377: 552 (1995); Chehab et al., Genes Dev. 14: 278 (2000); Harper et al., Mol. Biol. Cell 6: 387 (1995); Hirao et al., Science 287:1824 (2000); Shieh et al., Genes Dev. 14: 289(2000)).
Mutational loss of DNA damage checkpoint function is associated with, and is a contributory factor in, many cancers. See for example, McDonald et al. ER 3rd, El-Deiry W S. Checkpoint genes in cancer. Ann Med. 2001 March; 33(2):113-22; Wassmann K, Benezra R. Mitotic checkpoints: from yeast to cancer. Curr Opin Genet Dev. 2001 February; 11(1):83-90; Molinari M. Cell cycle checkpoints and their inactivation in human cancer. Cell Prolif. 2000 October; 33(5):261-74; Dasika G K, Lin S C, Zhao S, Sung P, Tomkinson A, Lee E Y. DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis. Oncogene. 1999 Dec. 20; 18(55):7883-99; and Nojima H. Cell cycle checkpoints, chromosome stability and the progression of cancer. Hum Cell. 1997 December; 10(4):221-30, each of which is incorporated by reference. Agents that selectively kill or retard the growth of checkpoint-deficient tissues while sparing normal tissues are potential cancer therapeutic agents.
Screens for potential cancer therapeutic agents have typically been performed using single cells in culture. For example, U.S. Pat. No. 5,972,640 describes methods for contacting cultured cells that are deficient in a particular mitotic checkpoint with candidate agents in an attempt to identify agents that selectively arrest the growth of DNA damage checkpoint deficient cells. The behavior of such individual cultured cells, however, frequently differs dramatically from the behavior of tissues in response to the same agent. For example, it is possible that agents with the ability to arrest the growth of checkpoint deficient cells in tissues are not able to arrest the growth of those same checkpoint deficient cells when removed from the tissue context. Similarly, it is possible that agents with the ability to arrest the growth of checkpoint-deficient cells in culture are not able to arrest the growth of those same checkpoint-deficient cells in tissues. Thus, screens for agents that arrest the growth of checkpoint-deficient cells in culture can frequently misclassify therapeutic agents.